: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
AB: One of the fundamental tenets of scientific progress is the reproducibility of experimental results, which is largely dependent on the availability of the identical reagents used in the original report. When original findings cannot be repeated, the quality, stability or performance of the reagents used in the experiments come into question. Research antibodies have come under particularly intense, and unfortunately well-deserved, criticism for frequently being the underlying reason for the failure to reproduce outcomes. Reports indicate that an estimated $800 million is wasted globally in researchers’ time, materials and money attempting to do science with unreliable or poorly characterized antibodies1. This growing dissatisfaction with the state of commercially available research antibodies has led to articles in many of the top biomedicine journals that demand increased validation of these reagents1-4. These sentiments are echoed in new requirements by various funding agencies for scientists to document how antibodies and other reagents have been verified for performance.
As an antibody manufacturer, GeneTex acknowledges that there are significant issues within the commercial research antibody industry. In fact, we accept our part in this and must face the resultant scrutiny. We have realized that without industry-accepted standards for research antibodies, GeneTex must be on the forefront of antibody production, quality assurance and validation.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
AB: As an antibody manufacturer with our own production facility and testing laboratory, GeneTex has over 20 years of experience validating antibodies. Our antigen affinity-purified polyclonal antibodies are routinely evaluated using Western blot, immunocytochemistry and immunohistochemistry. As with all polyclonal antibodies, there is the concern of batch-to-batch differences due to variations in the immune response of the host. Therefore, each subsequent lot must be retested in all applications and species. This is a time-consuming, costly and labor-intensive process, but is necessary to guarantee consistency in GeneTex’s polyclonal antibody products. A similarly rigorous screening process is applied to the selection of our mouse monoclonal antibody reagents.
In addition, given the reality that antibodies often perform best in an application-dependent fashion, obtaining convincing data for multiple applications and species with one antibody is often impossible. As a result, multiple antibodies to the same target are produced and validated for specific needs. At this time, RNAi-mediated knockdown validation is also utilized on an increasing scale.
As a complementary approach to validate our products, GeneTex also encourages researchers to submit “Star Reviews” where they evaluate our antibodies and provide independent user reviews. GeneTex also cites published papers where an antibody was used successfully.
GeneTex has more recently implemented a systematic analysis of our antibody reagents using more definitive means to confirm specificity. These efforts are encapsulated in our “Knockout/Knockdown Validation” program. Beginning with our most popular products, GeneTex is evaluating its antibodies through Western blot using cell lysates generated by either shRNA-mediated knockdown or CRISPR-mediated knockout of the target protein. In particular, GeneTex’s leadership has made a substantial commitment to incorporate CRISPR-mediated knockout validation as a central pillar of the QA protocol.
: Do you have any advice, recommendations and/or best practices scientists should follow when selecting and validating an antibody?
AB: When looking to purchase an antibody, scientists can increase their chances of buying the best product for their needs if they follow some simple suggestions. First, try not to let price dictate choice. Antibodies are expensive reagents, but low-priced antibodies often have little supportive data on company websites. And though it is always wise to shop around, it is generally better to spend more for a well-validated antibody as it will likely prove more economical in the long run by minimizing waste of researcher time, materials and money, not to mention by eliminating artifactual results. Second, researchers should carefully read the datasheet. Antibodies are frequently application- and species-dependent. This type of information is almost universally listed on datasheets. Researchers should verify that the application and species of interest to them have been directly tested. If one doesn’t see data supporting use in one’s intended application or species, a call to the company should be placed. In addition, there is usually information about the sequence of the immunogen used to generate the antibody. This can be crucial when trying to determine how specific an antibody will be for one member of a protein family with high sequence identity among its members. If it is not listed on the datasheet, a call to technical support can often help identify the immunogen sequence. Third, most scientists learn of reliable reagents through publications and word-of-mouth. It is advisable to check publications to see what antibody is being used to detect a given target. There are often specific monoclonal antibody clones or polyclonal antibody products that are widely accepted for detection of particular targets. Finally, always carefully review the vendor data and look for antibodies whose specificity has been validated through knockdown or knockout technologies.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
AB: The notion that there are “bad” antibodies on the market has been around since the dawn of the commercial antibody market, though it has only been very recently that there has been a concerted call for action on an industry-wide level. Publications in major journals, surveys and studies by nonprofit organizations, and scientific conferences have all contributed to expanding the general awareness of this issue and possible solutions.1-4 These have included, for example, better training and education of junior scientists with regard to research antibody selection and usage, standardization of antibody reagents through recombinant means, and proposals listing specific “conceptual pillars” for antibody evaluation, among others.1,2,4 Other more technical papers describe the use of mass spec in conjunction with immunoprecipitation or other techniques to evaluate antibody quality.3
Perhaps the major concern about addressing the antibody reproducibility issue is the sheer magnitude of the problem. Companies have thousands of antibody reagents in their catalogs, and new targets are identified each year. Attempts to evaluate all of these products through knockdown/knockout or other definitive approaches will require significant financial and manpower outlays. In addition, more specific testing is needed to assess the fidelity of antibodies directed to particular isoforms or post-translational modifications. Nevertheless, the benefits of being able to pare down one’s catalog and offer reagents with complete confidence, not to mention the fact that this is the future of the industry, will encourage companies to move forward. It will be a long process, but perhaps the IP/mass spec approaches being developed now will expedite this.
: What else is your company doing to address reproducibility?
AB: In addition to the strategies described previously, GeneTex is also taking a longer view to addressing the antibody reproducibility challenge. Consistent with the comments of Bradbury and Plückthun,1 GeneTex is currently evaluating different platforms for the production of recombinant antibodies. We are convinced that this is the direction that the commercial research antibody market is heading, and GeneTex views this as a goal to be pursued over the next several years.
Until that time, GeneTex will continue to expand our knockout/knockdown program. For our antibody reagents that are directed against post-translational modifications or against proteins that are not expressed in easily transfectable cell lines, we will continue to design individual plans for specificity assessment incorporating drug treatments, point mutants, peptide blocking and known positive and negative control cell lines. GeneTex will also further refine immunogen sequence selection so that our antibodies will be specific for particular isoforms or proteins from families with high sequence identity.
The industry is at a point where our customers are demanding better validation of the antibody reagents that are offered. Those companies that fail to meet these higher expectations will lose the trust of the research community and will not survive. At GeneTex, we accept this challenge and will pursue the strategies described here to continue to earn the business and loyalty of scientists who depend on reliable antibodies to move life sciences discovery forward.
References
- Bradbury, A. & Plückthun, A. Nature 518, 27-29 (2015).
- Uhlen, M. et al. Nature Methods 13, 823-827 (2016).
- Marcon, E. et al. Nature Methods 12, 725-731 (2015).
- Freedman, L.P. et al. BioTechniques 61, 16-18 (2016).
Author Bio: Alex Ball, M.D. attended the University of California, Irvine (UCI) where he got his B.S. in Biological Sciences. He obtained an M.D. degree from the University of Southern California School of Medicine, and subsequently completed residency training in Internal Medicine at California Pacific Medical Center in San Francisco, CA. He then did bench research at UCI for many years in chromosome dynamics before coming to GeneTex in 2012 as a senior scientist.